Molecules of the follistatin-related protein family and uses thereof

ABSTRACT

Novel FMCP polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated, full-length FMCP proteins, the invention further provides isolated FMCP fussion proteins, antigenic peptides and anti-FMCP antibodies. The invention also provides FMCP nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a FMCP gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

This application is a divisional application of Ser. No. 08/972,008filed on Nov. 17, 1997, now U.S. Pat. No. 5,942,420. The contents of allof the aforementioned application(s) are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

Follistatin is a single-chain glycoprotein of 35 kDa which is composedof four cysteine-rich domains, three of which are homologous and highlyconserved. (Lane et al. (1994) The FASEB Journal 8:163-173; Esch et al.(1987) Mol. Endo. 1:849-855; Sugano et al. (1994) Frontiers inEndocrinology Vol. 3: Inhibin and Inhibin-related Proteins, Rome:Ares-Serono Symposia, 69-80). Follistatin domains have recently beendescribed in several mosaic proteins, including agrin (Rupp et al.(1991) Neuron 6:811-823), osteonectin/SPARC (Lankat-Buttgereit et al.(1988) FEBS Lett. 236:352-356), and the brain-specific extracellularmatrix glycoprotein, SC1 (Johnston et al. (1990) Neuron 2: 165-176; seealso, Patthy et al. (1993) Trends Neurosci. 16:76-81). It has beenproposed that modules donated to mosaic proteins retain the functionthey had in the donor protein. (Eib et al. (1996) J. Neurochem. 67(3)1047-1055).

Follistatin binds the transforming growth factor-β (TGF-β) familymembers activin-A and inhibin. (Michel et al. (1993) Molecular andCellular Endocrinology 91:1-11). The family of TGF-β proteins includes,among others, activin-A and inhibin. (Eib et al. (1996) J. Neurochem.67:1047-1055). Members of the TGF-β family are multifunctional cytokineswith physiological effects on the growth and differentiation of avariety of normal and neoplastic cells (Sporn et al. (1992) J. Cell.Biol. 119:1017-1021). It has been proposed that follistatin and otherfollistatin-related molecules act by regulating the availability ofTGF-β-related and/or other growth factors thereby influencing cellularmigration, proliferation, and differentiation (Amthor (1996) Dev. Biol.178:343-361).

Follistatin and follistatin-related molecules have been found tomodulate a variety of biological processes. For example, follistatin hasbeen identified as a regulator of pituitary follice stimulating hormone(FSH) secretion (Ueno et al. (1990) Progress in Growth Factor Research2:113-124; Besecke et al. (1997) Endocrinology 138:2841-2848).Follistatins have also been characterized as growth factors (Vale et al.(1988) Recent Progress in Hormone Research 44:1-34; Link et al. (1997)Experimental Cell Research 233:350-362), and embryo modulators(Huylebroeck et al. (1994) Frontiers in Endocrinology, Vol. 3: Inhibinand Inhibin-related Proteins, Rome: Ares-Serono Symposia Publications,271-288; Petraglia (1996)). Osteonectin, which contains a single.follistatin domain, binds the platelet-derived growth factor (PDGF),preventing PDGF receptor activation (Raines et al. (1992) Proc. Natl.Acad. Sci. 89:1281-1304). Also, the follistatin domains in agrin havebeen reported to act in binding and thus creating local concentrationsof TGF-β family members in motor neurons and muscle (Patthy et al.(1993)). In addition, follistatin has high affinity for heparin sulfateside chains of membrane proteoglycans. (Nakamura et al. (1991) J. Biol.Chem. 266:19432-19437).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules with a follistatin-like domain, referred to herein as“Follistatin-Module-Containing-Protein” (FMCP) and nucleic acidmolecules. Thus, the presence of follistatin-related domains in aprotein indicates a role in the binding of molecules structurallyrelated to TGF-β family members (Eib et al. (1996) J. Neurochem.67:1047-1055). TGF-β superfamily members are multifunctional cytokineswhich modulate a number of functions. Therefore, the FMCP molecules ofthe present invention are useful as modulating agents in regulating avariety of cellular processes. Accordingly, in one aspect, thisinvention provides isolated nucleic acid molecules encoding FMCPproteins or biologically active portions thereof, as well as nucleicacid fragments suitable as primers or hybridization probes for thedetection of FMCP-encoding nucleic acids. In one embodiment, an isolatednucleic acid molecule of the present invention encodes a FMCP proteinwhich includes a follistatin cysteine-rich domain. In anotherembodiment, the FMCP nucleic acid molecule is a naturally occurringnucleotide sequence.

In another embodiment, a FMCP nucleic acid molecule is 45% homologous tothe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98546, or a complement thereof. In a preferredembodiment, an isolated FMCP nucleic acid molecule encodes the aminoacid sequence of human FMCP.

In another embodiment, a FMCP nucleic acid includes a nucleotidesequence encoding a protein having an amino acid sequence sufficientlyhomologous to a follistatin cysteine-rich domain amino acid sequence ofSEQ ID NO:2. In a preferred embodiment, a FMCP nucleic acid molecule hasthe nucleotide sequence shown SEQ ID NO:1, SEQ ID NO:3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98546.

In yet another preferred embodiment, a FMCP nucleic acid moleculeincludes a nucleotide sequence encoding a protein having an amino acidsequence at least 45% homologous to the amino acid sequence of SEQ IDNO:2.

Another embodiment of the invention features isolated FMCP proteinhaving an amino acid sequence 55% homologous to a follistatincysteine-rich domain of SEQ ID NO:2 (e.g., about amino acid residues97-243). Another embodiment of the invention features isolated FMCPprotein having an amino acid sequence at least about 65%, prefereably75%, 85%, or 95% homologous to a follistatin cysteine-rich domain of SEQID NO:2 (e.g., about amino acid residues 97-243). Yet another embodimentof the invention features isolated FMCP protein having an amino acidsequence at least about 55% homologous to the amino acid sequence of SEQID NO:4 or SEQ ID NO:5. Another embodiment of the invention featuresisolated FMCP protein having an amino acid sequence at least about 65%,preferably 75%, 85%, or 95% homologous to the amino acid sequence of SEQID NO:4 or SEQ ID NO:5.

Yet another embodiment of the invention features isolated FMCP proteinwhich is encoded by a nucleic acid molecule having a nucleotide sequenceat least about 55% homologous to a follistatin cysteine-rich domain ofSEQ ID NO:2 (e.g., about nucleotides 311 to 751 of SEQ ID NO:1). Anotherembodiment of the invention features isolated FMCP protein which isencoded by a nucleic acid molecule having a nucleotide sequence at leastabout 65%, preferably 75%, 85%, or 95% homologous to a follistatincysteine-rich domain of SEQ ID NO:2 (e.g., nucleotides 311 to 751 of SEQID NO:1). This invention further features isolated FMCP protein which isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1 (e.g., aboutnucleotides 311 to 751 of SEQ ID NO:1).

In another embodiment, an isolated nucleic acid molecule of the presentinvention encodes a FMCP protein which includes a signal sequence and issecreted. In another embodiment, an isolated nucleic acid molecule ofthe present invention encodes a FMCP protein which includes a signalsequence and is retained in an intracellular compartment. In anotherembodiment, the FMCP nucleic acid molecule encodes a FMCP protein and isa naturally occurring nucleotide sequence.

Another embodiment of the invention features FMCP nucleic acid moleculeswhich specifically detect FMCP nucleic acid molecules relative tonucleic acid molecules encoding other molecules with follistatin-likedomains. For example, in one embodiment, a FMCP nucleic acid moleculehybridizes under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence of nucleotides 23 to 811 of SEQ IDNO:1 as shown in SEQ ID NO:3. In another embodiment, the FMCP nucleicacid molecule is at least 500 nucleotides in length and hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98546, or a complement thereof.

In a preferred embodiment, an isolated FMCP nucleic acid moleculecomprises nucleotides 311-523 of SEQ ID NO:1 as shown in SEQ ID NO:4which encodes one follistatin cysteine-rich domain of FMCP, or acomplement thereof. In another preferred embodiment, an isolated FMCPnucleic acid molecule comprises nucleotides 533-751 of SEQ ID NO:1 asshown in SEQ ID NO:5 which encodes a second follistatin cysteine-richdomain of FMCP, or complement thereof. In another embodiment, a FMCPnucleic acid molecule further comprises nucleotides 1-523 of SEQ IDNO:1. In another embodiment, a FMCP nucleic acid molecule furthercomprises nucleotides 1-751. In yet another preferred embodiment, a FMCPnucleic acid molecule further comprises nucleotides 311-2525 of SEQ IDNO:1.

Another embodiment the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a FMCP nucleic acid.

Another aspect of the invention provides a vector comprising a FMCPnucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment the inventionprovides a host cell containing a vector of the invention. The inventionalso provides a method for producing FMCP protein by culturing in asuitable medium, a host cell of the invention containing a recombinantexpression vector such that FMCP protein is produced.

Another aspect of this invention features isolated or recombinant FMCPproteins and polypeptides. In one embodiment, an isolated FMCP proteinhas a follistatin cysteine-rich domain and is soluble or secreted orretained in an intracellular compartment and lacks a transmembrane orcytoplasmic domain. In another embodiment, an isolated FMCP protein hasan amino acid sequence sufficiently homologous to a follistatincysteine-rich domain amino acid sequence of SEQ ID NO:2. In a preferredembodiment, a FMCP protein has the amino acid sequence of SEQ ID NO:2.

Another embodiment of the invention features isolated FMCP proteinhaving an amino acid sequence at least about 45% homologous to the aminoacid sequence of SEQ ID NO:2. Another embodiment of the inventionfeatures isolated FMCP protein having an amino acid sequence at leastabout 55% homologous to the amino acid sequence of SEQ ID NO:2. Anotherembodiment of the invention features isolated FMCP protein having anamino acid sequence at least about 65% homologous to the amino acidsequence of SEQ ID NO:2. Another embodiment of the invention featuresisolated FMCP protein having an amino acid sequence at least about 75%homologous to the amino acid sequence of SEQ ID NO:2. Yet anotherembodiment of the invention features isolated FMCP protein having anamino acid sequence at least about 85% homologous to the amino acidsequence of SEQ ID NO:2. Yet another embodiment of the inventionfeatures isolated FMCP protein having an amino acid sequence at leastabout 95% homologous to the amino acid sequence of SEQ ID NO:2. Yetanother embodiment of the invention features isolated FMCP protein whichis encoded by a nucleic acid molecule having a nucleotide sequence atleast about 45% homologous to a nucleotide sequence of SEQ ID NO:1, SEQID NO:3, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98546 or a complement thereof.This invention further features isolated FMCP protein which is encodedby a nucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98546.

Another embodiment of the invention features isolated FMCP proteinhaving an amino acid sequence 55% homologous to a follistatincysteine-rich domain of SEQ ID NO:2 (e.g., about amino acid residues97-243). Another embodiment of the invention features isolated FMCPprotein having and amino acid sequence at least about 65%, prefereably75%, 85%, or 95% homologous to a follistatin cysteine-rich domain of SEQID NO:2 (e.g., about amino acid residues 97-243). Yet another embodimentof the invention features isolated FMCP protein having an amino acidsequence at least about 55% homologous to the amino acid sequence of SEQID NO:4 or SEQ ID NO:5. Another embodiment of the invention featuresisolated FMCP protein having an amino acid sequence at least about 65%,preferably 75%, 85%, or 95% homologous to the amino acid sequence of SEQID NO:4 or SEQ ID NO:5.

Yet another embodiment of the invention features isolated FMCP proteinwhich is encoded by a nucleic acid molecule having a nucleotide sequenceat least about 55% homologous to a follistatin cysteine-rich domain ofSEQ ID NO:2 (e.g., about nucleotides 311 to 751 of SEQ ID NO:1). Anotherembodiment of the invention features isolated FMCP protein which isencoded by a nucleic acid molecule having a nucleotide sequence at leastabout 65%, preferably 75%, 85%, or 95% homologous to a follistatincysteine-rich domain of SEQ ID NO:2 (e.g., nucleotides 311 to 751 of SEQID NO:1). This invention further features isolated FMCP protein which isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1 (e.g., aboutnucleotides 311 to 751 of SEQ ID NO:1).

The FMCP proteins of the present invention, or biologically activeportions thereof, can be operatively linked to a non-FMCP polypeptide toform FMCP fusion proteins. The invention further features antibodiesthat specifically bind FMCP proteins, such as monoclonal or polyclonalantibodies. In addition, the FMCP proteins or biologically activeportions thereof can be incorporated into pharmaceutical compositions,which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of FMCP activity or expression in a biological sample bycontacting the biological sample with an agent capable of detecting anindicator of FMCP activity such that the presence of FMCP activity isdetected in the biological sample.

In another aspect, the invention provides a method for modulating FMCPactivity comprising contacting the cell with an agent that modulatesFMCP activity such that FMCP activity in the cell is modulated. In oneembodiment, the agent inhibits FMCP activity. In another embodiment, theagent stimulates FMCP activity. In one embodiment, the agent is anantibody that specifically binds to FMCP protein. In another embodiment,the agent modulates expression of FMCP by modulating transcription of aFMCP gene or translation of a FMCP mRNA. In yet another embodiment, theagent is a nucleic acid molecule having a nucleotide sequence that isantisense to the coding strand of the FMCP mRNA or the FMCP gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant FMCP proteinor nucleic acid expression or activity by administering an agent whichis a FMCP modulator to the subject. In one embodiment, the FMCPmodulator is a FMCP protein. In another embodiment the FMCP modulator isa FMCP nucleic acid molecule. In yet another embodiment, the FMCPmodulator is a peptide, peptidomimetic, or other small molecule. In apreferred embodiment, the disorder characterized by aberrant FMCPprotein or nucleic acid expression is a proliferative or differentiativedisorder.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion characterized by at leastone of (i) aberrant modification or mutation of a gene encoding a FMCPprotein; (ii) mis-regulation of said gene; and (iii) aberrantpost-translational modification of a FMCP protein, wherein a wild-typeform of said gene encodes an protein with a FMCP activity.

In another aspect the invention provides a method for identifying acompound that binds to or modulates the activity of a FMCP protein, byproviding a indicator composition comprising a FMCP protein having FMCPactivity, contacting the indicator composition with a test compound, anddetermining the effect of the test compound on FMCP activity in theindicator composition to identify a compound that modulates the activityof a FMCP protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cDNA sequence and predicted amino acid sequence ofhuman FMCP (also referred to as “TANGO 91”). The nucleotide sequencecorresponds to nucleic acids 1 to 2525 of SEQ ID NO:1 which includes the5′ and 3′ untranslated regions or SEQ ID NO:3 which corresponds to theopen reading frame (nucleotides 23-811 of SEQ ID NO:1). The amino acidsequence of FMCP corresponds to amino acids 1 to 263 of SEQ ID NO:2.

FIG. 2 depicts an alignment of the amino acid sequences of human FMCP(corresponding to amino acids 97 to 243 of SEQ ID NO:2) and humanfollistatin cysteine-rich domain (Swiss-Prot™ Accession No. P19883).Alignment of the human FMCP protein with the human follistatin proteinusing Wisconsin GCG sequence alignment program GAP revealed that FMCP is43% identical and 61% similar to the human follistatin gene. Thisalignment included a Gap Weight of 3.0 and a Length Weight of 0.1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel moleculeshaving at least one follistatin cysteine-rich domain, referred to hereinas FMCP (Follistatin Module Containing Protein) and nucleic acidmolecules, which comprise a family of molecules having certain conservedstructural and functional features. The term “family” when referring tothe protein and nucleic acid molecules of the invention is intended tomean two or more proteins or nucleic acid molecules having a commonstructural domain and having sufficient amino acid or nucleotidesequence homology as defined herein. Such family members can benaturally occurring and can be from either the same or differentspecies. For example, a family can contain a first protein of humanorigin and a homologue of that protein of murine origin, as well as asecond, distinct protein of human origin and a murine homologue of thatprotein. Members of a family may also have common functionalcharacteristics.

In one embodiment, a FMCP family is identified based on the presence ofat least one “follistatin cysteine-rich domain” in the protein orcorresponding nucleic acid molecule. As used herein, the term“follistatin cysteine-rich domain” refers to a protein domain having anamino acid sequence of about 30 to 200 amino acid residues in length, ofwhich at least about 3 and up to about 20 amino acids are the amino acidresidue cysteine. More preferably, the follistatin cysteine-rich domainis at least about 40 to about 150 amino acid residues in length, ofwhich at least about 4 and up to about to 15 amino acids are the aminoacid cysteine. More preferably, the follistatin cysteine-rich domain isat least about 50 to about 130 amino acid residues in length, of whichat least about 5 and up to about 13 amino acids are the amino acidcysteine. More preferably, the follistatin cysteine-rich domain is atleast about 60 to 110 amino acid residues in length of which at leastabout 6 and up to about 11 amino acid residues are the amino acidcysteine. More preferably, the follistatin cysteine-rich domain is atleast about 70 to 90 amino acid residues in length of which at leastabout 7 and up to about 9 amino acid residues are the amino acidcysteine. Preferably, the follistatin cysteine-rich domain contains atleast 10 cysteine residues.

Preferably, the follistatin cysteine-rich domain of FMCP has cysteineresidues which are located in the domain in the same or similarpositions as cysteine residues in a follistatin cysteine-rich domain ofa related FMCP family member or FMCP homolog. For example, when a FMCPprotein of the invention is aligned with a FMCP family member or homologfor purposes of comparison (see e.g., FIG. 2) preferred cysteine-richdomains of the invention are those in which cysteine residues in theamino acid sequence of FMCP are located in the same or similar positionas the cysteine residues in the FMCP family member or FMCP homolog. Asan illustrative embodiment, FIG. 2 shows cysteine residues located inthe same or similar positions of the human follistatin protein and theFMCP protein at the following locations: amino acid number 95 of thehuman follistatin protein and amino acid number 99 of the FMCP protein;amino acid number 100 of the human follistatin protein and amino acidnumber 104 of the FMCP protein; and amino acid number 106 of the humanfollistatin protein and amino acid number 110 of the FMCP protein.

In another embodiment, a FMCP family is identified based on the presenceof at least one follastatin cysteine-rich domain in the protein orcorresponding nucleic acid molecule in which at least about 10-15% ofthe amino acid residues of the domain are cysteine residues.

In one embodiment, a FMCP protein includes a cysteine rich domain havingat least about 55%, preferably at least about 65%, and more preferablyabout 75%, 85%, or 95% amino acid sequence homology to a follistatincysteine-rich domain of SEQ ID NO:2. A preferred follistatincysteine-rich domain includes amino acid residues 97 to 243 of SEQ IDNO:2. In another embodiment, a follistatin cysteine-rich domain includesamino acid residues 97 to 167 of SEQ ID NO:2 (as shown in SEQ ID NO:4)or amino acid residues 171 to 243 of SEQ ID NO:2 (as shown in SEQ IDNO:5). Preferably, a FMCP protein includes at least two follistatincysteine-rich domains, more preferably at least three follistatincysteine-rich domains, and more preferably at least four or fivefollistatin cysteine-rich domains.

Preferred FMCP molecules of the present invention have an amino acidsequence sufficiently homologous to a follistatin cysteine-rich domainamino acid sequence of SEQ ID NO:2. As used herein, the term“sufficiently homologous” refers to a first amino acid or nucleotidesequence which contains a sufficient or minimum number of identical orequivalent (e.g., an amino acid residue which has a similar side chain)amino acid residues or nucleotides to a second amino acid or nucleotidesequence such that the first and second amino acid or nucleotidesequences have a common structural domain and/or common functionalactivity. For example, amino acid or nucleotide sequences which containa common structural domain having about 40% homology, preferably 50%homology, more preferably 60%-70% homology are defined herein assufficiently homologous. In one embodiment, the a FMCP protein containsa follistatin cysteine-rich domain and a FMCP activity.

As used interchangeably herein a “FMCP activity”, “biological activityof FMCP” or “functional activity of FMCP”, refers to an activity exertedby a FMCP protein, polypeptide or nucleic acid molecule on a FMCPresponsive cell as determined in vivo, or in vitro, according tostandard techniques. In one embodiment, a FMCP activity is a directactivity, such as an association with or an enzymatic activity on asecond protein. In another embodiment, a FMCP activity is an indirectactivity, such as a cellular signaling activity mediated by interactionof the FMCP protein with a second protein. In a preferred embodiment, aFMCP activity includes at least one or more of the following activities:(i) complex formation between a FMCP protein and a cytokine; (ii)interaction of a FMCP protein with a protein having substantial homologyto the TGF-β family of proteins; (iii) interaction of a FMCP proteinwith a TGF-β family member protein; and (iv) interaction of a FMCPprotein with other proteins. In yet another preferred embodiment, a FMCPactivity is at least one or more of the following activities: (i)modulation of TGF-β-related protein activity; (ii) regulation ofcellular proliferation; (iii) regulation of cellular differentiation;and (iv) regulation of cell survival.

Accordingly, another embodiment of the invention features isolated FMCPproteins and polypeptides having a FMCP activity. Preferred FMCPproteins have at least one follistatin cysteine-rich domain (andpreferably two or more follistatin cysteine-rich domains) and a FMCPactivity. In another preferred embodiment, the FMCP protein has at leastone follistatin cysteine-rich domain (and preferably two or morefollistatin cysteine-rich domains), a FMCP activity and an amino acidsequence sufficiently homologous to an amino acid sequence of SEQ IDNO:2.

Accordingly, in one embodiment, FMCP proteins of the invention containat least one follistatin cysteine-rich domain (and preferably two ormore follistatin cysteine-rich domains) and have an amino acid sequencesufficiently homologous to amino acid sequence of SEQ ID NO:2. Inanother preferred embodiment, the FMCP protein has at least onefollistatin cysteine-rich domain (and preferably two or more follistatincysteine-rich domains), an amino acid sequence sufficiently homologousto amino acid sequence of SEQ ID NO:2 and a FMCP activity.

Yet another embodiment of the invention features FMCP molecules whichcontain a signal sequence. As used herein, a “signal sequence” refers toa peptide containing about 20 amino acids which occurs at the extremeN-terminal end of secretory and integral membrane proteins and whichcontains large numbers of hydrophobic amino acid residues. Such a“signal sequence”, also referred to in the art as a “signal peptide”,serves to direct a protein containing such a sequence to a lipidbilayer.

In a particularly preferred embodiment, the FMCP protein and nucleicacid molecules of the present invention are human FMCP molecules. Anucleotide sequence of a human FMCP protein is shown in FIG. 1 and inSEQ ID NO:1, SEQ ID NO:3, and the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 98546. Apredicted amino acid sequence of the isolated human FMCP protein isshown in FIG. 1 and in SEQ ID NO:2. In addition, the nucleotide sequencecorresponding to the coding region of the human FMCP cDNA (nucleotides23-811) is represented as SEQ ID NO:3.

The human FMCP cDNA, which is approximately 2525 nucleotides in lengthincluding untranslated regions as indicated in SEQ ID NO:1, or whichcorresponds to the open reading frame as indicated in SEQ ID NO:3,encodes a protein having a molecular weight of approximately 25 kDa(excluding post-translational modifications) and which is approximately263 amino acid residues in length. The human FMCP protein contains twofollistatin cysteine-rich domains. A FMCP follistatin cysteine-richdomain can be found at least, for example, from about amino acids 97-167of SEQ ID NO:2 (Asp97 to Cys167 of the human FMCP amino acid sequence)and, for example, from about amino acids 171-243 of SEQ ID NO:2 (Cys171to Cys243 of the human FMCP amino acid sequence). These regions containamino acid sequences of which at least about 10% of the total amino acidresidues are cysteine residues and are located in the same or similarpositions as the cysteien residues of the a FMCP homolog, e.g., humanfollistatin. The human FMCP protein is a secreted protein which lacks atransmembrane domain.

Alignment of the human FMCP protein with the human follistatin proteinusing Wisconsin GCG sequence alignment program GAP revealed that FMCP is43% identical and 61% similar to the human follistatin protein.Similarly, when FMCP is aligned with the human follistatin-related gene(hFRP) (Swiss Prot Q12841) using GAP, it shows 24% identity and 48%similarity to hFRP.

Alignment of the follistatin domains of the human FMCP protein (as shownin SEQ ID NO:4 and SEQ ID NO:5) with the follistatin domains of thehuman follistatin protein using DNASTAR MegAlign alignment program ofLipman-Pearson using a ktuple of 2, a gap penalty of 4, and a gap lengthpenalty of 12, revealed that both follistatin cysteine-rich domains ofthe FMCP protein are 55% homologous to the human follistatin domains.

A 2.5 kb FMCP mRNA transcript is expressed in human tissues includingheart, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,prostrate, testis, ovary, small intestine, and colon, with morepronounced expression observed in human placenta, testis, and heart. Inaddition, a smaller FMCP transcript of approximately 1.4 kb is found inheart, placenta, lung, kidney, and testis.

A GenBank™ search using the human FMCP nucleotide sequence of SEQ IDNO:1, revealed eleven EST sequences, two human, eight mouse, and onerat, which were at least 80% identical to different regions of thenucleotide sequence of SEQ ID NO:1. The EST sequences having greaterthan 80% identity are listed in Table 1, as well as, the nucleotides ofSEQ ID NO:1 to which each EST sequence corresponds. Unless specifiedotherwise, all EST sequences are annotated.

TABLE 1 corresponding nucleotides of human FMCP (SEQ ID NO:1 nucleotidesOR SEQ ID % Accession No. SPECIES of EST NO:3) Identity AA020306 mouse97 > 461  62-426 84 AA015105 mouse 76 > 365  62-351 85 W18317 mouse 78 >343  62-327 85 W14649 mouse  1 > 249 165-415 86 AA051472 mouse 77 > 327 62-312 84 AA408816 mouse 35 > 263 489-717 91 D31566 human 24 > 162598-765 96 (not annotated) AA023955 mouse  3 < 179 577-697 84 H32687 rat 1 > 176 123-298 82 AA552990 human  1 < 174 581-775 89 AA020355 mouse23 > 173 284-434 85

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode FMCP proteins or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify FMCP-encoding nucleic acids (e.g., FMCP mRNA) andfragments for use as PCR primers for the amplification or mutation ofFMCP nucleic acid molecules. As used herein, the term “nucleic acidmolecule” is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, the isolated FMCP nucleic acid molecule can containless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule ingenomic DNA of the cell from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture mediumwhen produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number 98546, or a complement of any of thesenucleotide sequences, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all orportion of the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:3, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number 98546 as a hybridization probe, FMCP nucleicacid molecules can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook, J., Fritsh, E. F., andManiatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to FMCP nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:3, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98546. The sequence of SEQ IDNO:1, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number 98546 corresponds to thehuman FMCP cDNA. These cDNA comprise sequences encoding the human FMCPprotein (i.e., “the coding region”, from nucleotides 23 to 811 of SEQ IDNO:1), as well as 5′ untranslated sequences (nucleotides 1 to 22) and 3′untranslated sequences (nucleotides 812 to 2525 of SEQ ID NO:1).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98546, or a portion of this nucleotide sequence. Anucleic acid molecule which is complementary to the nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98546 isone which is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98546 suchthat it can hybridize to the nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98546 thereby forming a stableduplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 60-65%, preferably at least about 70-75%, more preferable atleast about 80-85%, and even more preferably at least about 90-95% ormore homologous to the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:3, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98546, or a portion of thisnucleotide sequence.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98546, for example, a fragment which can be used asa probe or primer or a fragment encoding a biologically active portionof FMCP. The nucleotide sequence determined from the cloning of thehuman FMCP gene allows for the generation of probes and primers designedfor use in identifying and/or cloning FMCP homologues in other celltypes, e.g. from other tissues, as well as FMCP homologues from othermammals. The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 100, 150,200, 250, 300, 350 or 400 consecutive nucleotides of SEQ ID NO:1, SEQ IDNO:3, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98546 sense, of an anti-sensesequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number 98546,or of a naturally occurring mutant of SEQ ID NO:1, SEQ ID NO:3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98546.

Probes based on the human FMCP nucleotide sequence can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a FMCP protein, such as by measuring a level ofa FMCP-encoding nucleic acid in a sample of cells from a subject e.g.,detecting FMCP mRNA levels or determining whether a genomic FMCP genehas been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of FMCP”can be prepared by isolating a portion of SEQ ID NO:1, SEQ ID NO:3, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number 98546 which encodes a polypeptide having a FMCPbiological activity (the biological activities of the FMCP proteins havepreviously been described), expressing the encoded portion of FMCPprotein (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of FMCP. For example, a nucleic acidfragment encoding a biologically active portion of FMCP includes afollistatin cysteine-rich domain, e.g., amino acid residues 97-243 ofSEQ ID NO:2. In another embodiment, a nucleic acid fragment encoding abiologically active portion of FMCP includes a follistatin cysteine-richdomain, e.g., SEQ ID NO:4 or SEQ ID NO:5. In another embodiment, anucleic acid fragment encoding a biologically active portion of FMCPincludes a follistatin cysteine-rich domain includes the DNA encodingsuch domains, e.g., at least nucleic acids 311-523 of SEQ ID NO:1 whichencodes the human FMCP follistatin domain represented by amino acidresidues 97-167 of SEQ ID NO:2 (as shown in SEQ ID NO:4) or at leastnucleic acids 533-751 of SEQ ID NO:1 which encodes the human FMCPfollistatin domain represented by amino acid residues 171-243 of SEQ IDNO:2 (as shown in SEQ ID NO:5).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98546 due to degeneracy of the genetic code and thusencode the same FMCP protein as that encoded by the nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98546. Inanother embodiment, an isolated nucleic acid molecule of the inventionhas a nucleotide sequence encoding a protein having an amino acidsequence shown in SEQ ID NO:2.

In addition to the human FMCP nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98546, it will be appreciated bythose skilled in the art that DNA sequence polymorphisms that lead tochanges in the amino acid sequences of FMCP may exist within apopulation (e.g., the human population). Such genetic polymorphism inthe FMCP gene may exist among individuals within a population due tonatural allelic variation. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules comprising an openreading frame encoding a FMCP protein, preferably a mammalian FMCPprotein. Such natural allelic variations can typically result in 1-5%variance in the nucleotide sequence of the FMCP gene. Any and all suchnucleotide variations and resulting amino acid polymorphisms in FMCPthat are the result of natural allelic variation and that do not alterthe functional activity of FMCP are intended to be within the scope ofthe invention.

Moreover, nucleic acid molecules encoding FMCP proteins from otherspecies, and thus which have a nucleotide sequence which differs fromthe human sequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98546 are intended to be within the scope of theinvention. Nucleic acid molecules corresponding to natural allelicvariants and homologues of the FMCP cDNAs of the invention can beisolated based on their homology to the human FMCP nucleic acidsdisclosed herein using the human cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. For example, a soluble human FMCPcDNA can be isolated based on its homology to human membrane-bound FMCP.Likewise, a membrane-bound human FMCP cDNA can be isolated based on itshomology to soluble human FMCP.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98546. In another embodiment, the nucleic acid is atleast 30, 50, 100, 250 or 500 nucleotides in length. In anotherembodiment, an isolated nucleic acid molecule of the inventionhybridizes to the coding region. As used herein, the term “hybridizesunder stringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%homologous to each other typically remain hybridized to each other.Preferably, the conditions are such that sequences at least about 65%,more preferably at least about 70%, and even more preferably at leastabout 75% homologous to each other typically remain hybridized to eachother. Such stringent conditions are known to those skilled in the artand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleicacid molecule of the invention that hybridizes under stringentconditions to the sequence of SEQ ID NO:1, SEQ ID NO:3, corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

In addition to naturally-occurring allelic variants of the FMCP sequencethat may exist in the population, the skilled artisan will furtherappreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, thereby leading tochanges in the amino acid sequence of the encoded FMCP protein, withoutaltering the functional ability of the FMCP protein. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98546. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of FMCP (e.g., the sequence of SEQ ID NO:2)without altering the biological activity, whereas an “essential” aminoacid residue is required for biological activity. For example, aminoacid residues that are conserved among the FMCP proteins of the presentinvention, as well as, among the follistatin family of proteins (asindicated by the alignment presented as FIG. 2) are predicted to beparticularly unamenable to alteration.

For example, preferred FMCP proteins of the present invention, containat least one follistatin cysteine-rich domain which are typicallyconserved regions in FMCP family members and FMCP homologs. As such,these conserved domains are not likely to be amenable to mutation. Otheramino acid residues, however, (e.g., those that are not conserved oronly semi-conserved among members of the follistatin proteins) may notbe essential for activity and thus are likely to be amenable toalteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding FMCP proteins that contain changes in amino acidresidues that are not essential for activity. Such FMCP proteins differin amino acid sequence from SEQ ID NO:2 yet retain biological activity.In one embodiment, the isolated nucleic acid molecule comprises anucleotide sequence encoding a protein, wherein the protein comprises anamino acid sequence at least about 45% homologous to the amino acidsequence of SEQ ID NO:2. Preferably, the protein encoded by the nucleicacid molecule is at least about 60% homologous to SEQ ID NO:2, morepreferably at least about 70% homologous to SEQ ID NO:2, more preferablyat least about 80% homologous to SEQ ID NO:2, even more preferably atleast about 90% homologous to SEQ ID NO:2, and most preferably at leastabout 95% homologous to SEQ ID NO:2.

An isolated nucleic acid molecule encoding a FMCP protein homologous tothe protein of SEQ ID NO:2 can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number 98546such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introducedinto SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98546 bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in FMCP is preferably replacedwith another amino acid residue from the same side chain family.Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a FMCP coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forFMCP biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:1, SEQ ID NO:3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98546, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

In a preferred embodiment, a mutant FMCP protein can be assayed for (1)the ability to form protein:protein interactions with otherfollistatin-related proteins, other cell-surface proteins, orbiologically active portions thereof; (2) complex formation between amutant FMCP protein and a FMCP ligand; (3) the ability of a mutant FMCPprotein to bind to an intracellular target protein or biologicallyactive portion thereof; (e.g. avidin proteins). In yet another preferredembodiment, a mutant FMCP can be assayed for the ability to performTGF-β super family member activities, such as, (i) complex formationbetween a FMCP protein and a cytokine; (ii) interaction of a FMCPprotein with a protein having substantial homology to the TGF-β familyof proteins; (iii) interaction of a FMCP protein with a TGF-β familymember protein; and (iv) interaction of a FMCP protein with otherproteins. In yet another preferred embodiment, a FMCP activity is atleast one or more of the following activities: (i) modulation ofTGF-β-related protein activity; (ii) regulation of cellularproliferation; (iii) regulation of cellular differentiation; and (iv)regulation of cell survival.

In addition to the nucleic acid molecules encoding FMCP proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire FMCP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding FMCP. Theterm “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the coding region of human FMCP corresponds to nucleotides 23-811 of SEQID NO:1, as shown in SEQ ID NO:3). In another embodiment, the antisensenucleic acid molecule is antisense to a “noncoding region” of the codingstrand of a nucleotide sequence encoding FMCP. The term “noncodingregion” refers to 5′ and 3′ sequences which flank the coding region thatare not translated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences encoding FMCP disclosed herein (e.g.,SEQ ID NO:1 or SEQ ID NO:3), antisense nucleic acids of the inventioncan be designed according to the rules of Watson and Crick base pairing.The antisense nucleic acid molecule can be complementary to the entirecoding region of FMCP mRNA, but more preferably is an oligonucleotidewhich is antisense to only a portion of the coding or noncoding regionof FMCP mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofFMCP mRNA. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a FMCP proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An a-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveFMCP mRNA transcripts to thereby inhibit translation of FMCP mRNA. Aribozyme having specificity for a FMCP-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a FMCP cDNA disclosedherein (i.e., SEQ ID NO:1, SEQ ID NO:3,). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a FMCP-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,FMCP mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, FMCP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the FMCP(e.g., the FMCP promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the FMCP gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

In preferred embodiments, the nucleic acids of FMCP can be modified atthe base moiety, sugar moiety or phosphate backbone to improve, e.g.,the stability, hybridization, or solubility of the molecule. Forexample, the deoxyribose phosphate backbone of the nucleic acids can bemodified to generate peptide nucleic acids (see Hyrup B. et al. (1996)Bioorganic & Medicinal Chemistry 4 (1):5-23). As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup B. et al.(1996) supra; Perry-O'Keefe et al. PNAS 93: 14670-675.

PNAs of FMCP can be used therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofFMCP can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as ‘artificialrestriction enzymes when used in combination with other enzymes, e.g.,S1 nucleases (Hyrup B. (1996) supra); or as probes or primers for DNAsequence and hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefesupra).

In another embodiment, PNAs of FMCP can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of FMCP can be generated which may combinethe advantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNAse H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup B. (1996) supra).The synthesis of PNA-DNA chimeras can be performed as described in HyrupB. (1996) supra and Finn P. J. et al. (1996) Nucleic Acids Research 24(17):3357-63. For example, a DNA chain can be synthesized on a solidsupport using standard phosphoramidite coupling chemistry and modifiednucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used as a between the PNA and the 5′ end of DNA(Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn P. J. et al. (1996) supra).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser, K. H. et al. (1975) BioorganicMed. Chem. Lett. 5: 1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. W088/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. W089/10134,published Apr. 25, 1988). In addition, oligonucleotides can be modifiedwith hybridization-triggered cleavage agents (See, e.g., Krol et al.,1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon,1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, etc.

II. Isolated FMCP Proteins and Anti-FMCP Antibodies

One aspect of the invention pertains to isolated FMCP proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-FMCP antibodies. In oneembodiment, native FMCP proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, FMCP proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a FMCP protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theFMCP protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of FMCPprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of FMCP protein having less than about 30% (by dryweight) of non-FMCP protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-FMCP protein,still more preferably less than about 10% of non-FMCP protein, and mostpreferably less than about 5% non-FMCP protein. When the FMCP protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of FMCP protein in which the protein isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of FMCP protein having less than about 30% (by dry weight)of chemical precursors or non-FMCP chemicals, more preferably less thanabout 20% chemical precursors or non-FMCP chemicals, still morepreferably less than about 10% chemical precursors or non-FMCPchemicals, and most preferably less than about 5% chemical precursors ornon-FMCP chemicals.

Biologically active portions of a FMCP protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the FMCP protein, e.g., the amino acidsequence shown in SEQ ID NO:2, which include less amino acids than thefull length FMCP proteins, and exhibit at least one activity of a FMCPprotein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the FMCP protein. A biologicallyactive portion of a FMCP protein can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length.

In one embodiment, a biologically active portion of a FMCP proteincomprises at least one follistatin cysteine-rich domain characteristicof the follistatin family of proteins.

It is to be understood that a preferred biologically active portion of aFMCP protein of the present invention may contain at least one of theabove-identified structural domains. A more preferred biologicallyactive portion of a FMCP protein may contain at least two of theabove-identified structural domains. An even more preferred biologicallyactive portion of a FMCP protein may contain at least three of theabove-identified structural domains. A particularly preferredbiologically active portion of a FMCP protein of the present inventionmay contain at least four of the above-identified structural domains. Amore particularly preferred biologically active portion of a FMCPprotein may have at least five of the above-identified structuraldomains. Finally, a most preferred biologically active portion of a FMCPprotein may contain at least six of the above-identified structuraldomains.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native FMCPprotein.

In a preferred embodiment, the FMCP protein has an amino acid sequenceshown in SEQ ID NO:2. In other embodiments, the FMCP protein issubstantially homologous to SEQ ID NO:2 and retains the functionalactivity of the protein of SEQ ID NO:2 yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail in subsection II below. Accordingly, in another embodiment,the FMCP protein is a protein which comprises an amino acid sequence atleast about 45% homologous to the amino acid sequence of SEQ ID NO:2 andretains the functional activity of the FMCP proteins of SEQ ID NO:2. Inanother embodiment, the FMCP protein is a protein having an amino acidsequence 55% homologous to a follistatin cysteine-rich domain of SEQ IDNO:2 (e.g., about amino acid residues 97-243, amino acid residues97-167, or amino acid residues 171-243). Another embodiment of theinvention features isolated FMCP protein having and amino acid sequenceat least about 65%, prefereably 75%, 85%, or 95% homologous to afollistatin cysteine-rich domain of SEQ ID NO:2 (e.g., about amino acidresidues 97-243). Yet another embodiment of the invention featuresisolated FMCP protein having an amino acid sequence at least about 55%homologous to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.Another embodiment of the invention features isolated FMCP proteinhaving an amino acid sequence at least about 65%, preferably 75%, 85%,or 95% homologous to the amino acid sequence of SEQ ID NO:4 or SEQ IDNO:5. In a preferred embodiment, the FMCP protein retains the functionalactivity of the FMCP proteins of SEQ ID NO:2.

Yet another embodiment of the invention features isolated FMCP proteinwhich is encoded by a nucleic acid molecule having a nucleotide sequenceat least about 55% homologous to a follistatin cysteine-rich domain ofSEQ ID NO:2 (e.g., about nucleotides 311 to 751 of SEQ ID NO:1). Anotherembodiment of the invention features isolated FMCP protein which isencoded by a nucleic acid molecule having a nucleotide sequence at leastabout 65%, preferably 75%, 85%, or 95% homologous to a follistatincysteine-rich domain of SEQ ID NO:2 (e.g., nucleotides 311 to 751 of SEQID NO:1). This invention further features isolated FMCP protein which isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1 (e.g., aboutnucleotides 311 to 751 of SEQ ID NO:1).

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”). The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=#ofidentical positions/total #of positions×100).

The invention also provides FMCP chimeric or fusion proteins. As usedherein, a FMCP “chimeric protein” or “fusion protein” comprises a FMCPpolypeptide operatively linked to a non-FMCP polypeptide. A “FMCPpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to FMCP, whereas a “non-FMCP polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the FMCP protein, e.g., aprotein which is different from the FMCP protein and which is derivedfrom the same or a different organism. Within a FMCP fusion protein theFMCP polypeptide can correspond to all or a portion of a FMCP protein.In a preferred embodiment, a FMCP fusion protein comprises at least onebiologically active portion of a FMCP protein. In another preferredembodiment, a FMCP fusion protein comprises at least two biologicallyactive portions of a FMCP protein. In another preferred embodiment, aFMCP fusion protein comprises at least three biologically activeportions of a FMCP protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the FMCP polypeptideand the non-FMCP polypeptide are fused in-frame to each other. Thenon-FMCP polypeptide can be fused to the N-terminus or C-terminus of theFMCP polypeptide.

For example, in one embodiment a FMCP fusion protein comprises a FMCPfollistatin cystein-rich domain domain operably linked to theextracellular domain of a second protein known to be involved incytokine activity. Such fusion proteins can be further utilized inscreening assays for compounds which modulate FMCP activity (such assaysare described in detail below).

In yet another embodiment, the fusion protein is a GST-FMCP fusionprotein in which the FMCP sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant FMCP.

In another embodiment, the fusion protein is a FMCP protein containing aheterologous signal sequence at its N-terminus. For example, the nativeFMCP signal sequence (i.e., about amino acids 1 to 26 of SEQ ID NO:2)can be removed and replaced with a signal sequence from another protein.In certain host cells (e.g., mammalian host cells), expression and/orsecretion of FMCP can be increased through use of a heterologous signalsequence.

In yet another embodiment, the fusion protein is a FMCP-immunoglobulinfusion protein in which the FMCP sequences comprising primarily thefollistatin cysteine-rich domains are fused to sequences derived from amember of the immunoglobulin protein family. The FMCP-immunoglobulinfusion proteins of the invention can be incorporated into pharmaceuticalcompositions and administered to a subject to inhibit an interactionbetween a FMCP ligand and a FMCP protein on the surface of a cell, tothereby suppress FMCP-mediated signal transduction in vivo. TheFMCP-immunoglobulin fusion proteins can be used to affect thebioavailability of a FMCP cognate ligand. Inhibition of the FMCPligand/FMCP interaction may be useful therapeutically for both thetreatment of proliferative and differentiative disorders, as well asmodulating (e.g. promoting or inhibiting) cell survival. Moreover, theFMCP-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-FMCP antibodies in a subject, to purify FMCPligands and in screening assays to identify molecules which inhibit theinteraction of FMCP with a FMCP ligand.

Preferably, a FMCP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AFMCP-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the FMCP protein.

The present invention also pertains to variants of the FMCP proteinswhich function as either FMCP agonists (mimetics) or as FMCPantagonists. Variants of the FMCP protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the FMCPprotein. An agonist of the FMCP protein can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of the FMCP protein. An antagonist of the FMCP proteincan inhibit one or more of the activities of the naturally occurringform of the FMCP protein by, for example, competitively binding to adownstream or upstream member of a cellular signaling cascade whichincludes the FMCP protein. Thus, specific biological effects can beelicited by treatment with a variant of limited function. In oneembodiment, treatment of a subject with a variant having a subset of thebiological activities of the naturally occurring form of the protein hasfewer side effects in a subject relative to treatment with the naturallyoccurring form of the FMCP proteins.

In one, variants of the FMCP protein which function as either FMCPagonists (mimetics) or as FMCP antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of the FMCP protein for FMCP protein agonist or antagonist activity. Inone embodiment, a variegated library of FMCP variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of FMCP variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential FMCP sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of FMCP sequences therein. There are avariety of methods which can be used to produce libraries of potentialFMCP variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential FMCP sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477.

In addition, libraries of fragments of the FMCP protein coding sequencecan be used to generate a variegated population of FMCP fragments forscreening and subsequent selection of variants of a FMCP protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a FMCP coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal and internalfragments of various sizes of the FMCP protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of FMCP proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recrusive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify FMCP variants (Arkin and Yourvan (1992) PNAS 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated FMCP library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily responds to aparticular cytokine in a FMCP-dependent manner. The transfected cellsare then contacted with the cytokine and the effect of expression of themutant on signaling by the cytokine can be detected, e.g. by measuringNF-κB activity or cell survival. Plasmid DNA can then be recovered fromthe cells which score for inhibition, or alternatively, potentiation ofcytokine induction, and the individual clones further characterized.

An isolated FMCP protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind FMCP using standardtechniques for polyclonal and monoclonal antibody preparation. Thefull-length FMCP protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of FMCP for use as immunogens. Theantigenic peptide of FMCP comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2 and encompasses an epitopeof FMCP such that an antibody raised against the peptide forms aspecific immune complex with FMCP. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofFMCP that are located on the surface of the protein, e.g., hydrophilicregions. A hydrophobicity analysis of the human FMCP protein sequenceindicates that the regions between amino acids 135-175 and 240-260 areparticularly hydrophilic and, therefore, are likely to encode surfaceresidues useful for targeting antibody production.

A FMCP immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed FMCP protein or a chemicallysynthesized FMCP polypeptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic FMCP preparation induces a polyclonal anti-FMCP antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-FMCPantibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as FMCP.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind FMCP. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of FMCP. A monoclonal antibody composition thustypically displays a single binding affinity for a particular FMCPprotein with which it immunoreacts.

Polyclonal anti-FMCP antibodies can be prepared as described above byimmunizing a suitable subject with a FMCP immunogen. The anti-FMCPantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized FMCP. If desired, the antibody moleculesdirected against FMCP can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-FMCP antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem . 255:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75),the more recent human B cell hybridoma technique (Kozbor et al. (1983)Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985),Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96)or trioma techniques. The technology for producing monoclonal antibodyhybridomas is well known (see generally R. H. Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med.,54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36).Briefly, an immortal cell line (typically a myeloma) is fused tolymphocytes (typically splenocytes) from a mammal immunized with a FMCPimmunogen as described above, and the culture supernatants of theresulting hybridoma cells are screened to identify a hybridoma producinga monoclonal antibody that binds FMCP.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-FMCP monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, YaleJ. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindFMCP, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-FMCP antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with FMCP to thereby isolateimmunoglobulin library members that bind FMCP. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol.Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram etal. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137;Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature(1990) 348:552-554.

Additionally, recombinant anti-FMCP antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)PNAS 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun etal. (1987) PNAS 84:214-218; Nishimura et al. (1987) Canc. Res.47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al.(1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985)Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S.Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan etal. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

An anti-FMCP antibody (e.g., monoclonal antibody) can be used to isolateFMCP by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-FMCP antibody can facilitate thepurification of natural FMCP from cells and of recombinantly producedFMCP expressed in host cells. Moreover, an anti-FMCP antibody can beused to detect FMCP protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the FMCP protein. Anti-FMCP antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding FMCP (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., FMCP proteins, mutant forms ofFMCP, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of FMCP in prokaryotic or eukaryotic cells. For example, FMCPcan be expressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the FMCP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(In Vitrogen Corp, San Diego, Calif.).

Alternatively, FMCP can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., Sf 9 cells) include the pAcseries (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to FMCP mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, FMCPprotein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning. A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding FMCP or can be introduced on a separate vector.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) FMCP protein.Accordingly, the invention further provides methods for producing FMCPprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding FMCP has been introduced) in asuitable medium such that FMCP protein is produced. In anotherembodiment, the method further comprises isolating FMCP from the mediumor the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichFMCP-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous FMCPsequences have been introduced into their genome or homologousrecombinant animals in which endogenous FMCP sequences have beenaltered. Such animals are useful for studying the function and/oractivity of FMCP and for identifying and/or evaluating modulators ofFMCP activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, a “homologous recombinant animal” is a non-humananimal, preferably a mammal, more preferably a mouse, in which anendogenous FMCP gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducingFMCP-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The humanFMCP cDNA sequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98546 can be introduced as a transgene into the genomeof a non-human animal. Alternatively, a nonhuman homologue of the humanFMCP gene, such as a mouse FMCP gene, can be isolated based onhybridization to the human FMCP cDNA (described further in subsection Iabove) and used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to the FMCP transgene to direct expression ofFMCP protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the FMCP transgene in its genomeand/or expression of FMCP mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding FMCP can further be bred to other transgenic animalscarrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a FMCP gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the FMCP gene. The FMCP gene can be a human gene(e.g., the cDNA of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98546), but more preferably, is a non-human homologue of a human FMCPgene. For example, a mouse homologue of human FMCP gene of SEQ ID NO:1,SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98546 can be used to construct ahomologous recombination vector suitable for altering an endogenous FMCPgene in the mouse genome. In a preferred embodiment, the vector isdesigned such that, upon homologous recombination, the endogenous FMCPgene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thevector can be designed such that, upon homologous recombination, theendogenous FMCP gene is mutated or otherwise altered but still encodesfunctional protein (e.g., the upstream regulatory region can be alteredto thereby alter the expression of the endogenous FMCP protein). In thehomologous recombination vector, the altered portion of the FMCP gene isflanked at its 5′ and 3′ ends by additional nucleic acid of the FMCPgene to allow for homologous recombination to occur between theexogenous FMCP gene carried by the vector and an endogenous FMCP gene inan embryonic stem cell. The additional flanking FMCP nucleic acid is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see e.g., Thomas, K. R.and Capecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedFMCP gene has homologously recombined with the endogenous FMCP gene areselected (see e.g., Li, E. et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec etal.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; andWO 93/04169 by Berns et al.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.Another example of a recombinase system is the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355.If a cre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein are required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

IV. Pharmaceutical Compositions

The FMCP nucleic acid molecules, FMCP proteins, and anti-FMCP antibodies(also referred to herein as “active compounds”) of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a FMCP protein or anti-FMCP antibody) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

Proteins containing follistatin domains are known to bind TGF-βsuperfamily members. TGF-β superfamily members are multifunctionalcytokines which modulate a number of functions. The nucleic acidmolecules, proteins, protein homologues, and antibodies described hereinwhich include follistatin-related domains, therefore, can be used in oneor more of the following methods: a) screening assays; b) detectionassays (e.g., chromosomal mapping, tissue typing, forensic biology), c)predictive medicine (e.g., diagnostic assays, prognostic assays,monitoring clinical trials, and pharmacogenomics); and d) methods oftreatment (e.g., therapeutic and prophylactic). As described herein, inone embodiment, a FMCP protein of the invention has the ability to bindand inactivate TGF-β family members. A FMCP protein interacts with othercellular proteins and can thus be used to (i) modulation ofTGF-β-related protein activity; (ii) regulation of cellularproliferation; (iii) regulation of of cellular differentiation; and (iv)regulation of cell survival. The isolated nucleic acid molecules of theinvention can be used to express FMCP protein (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect FMCP mRNA (e.g., in a biological sample) or a genetic lesion in aFMCP gene, and to modulate FMCP activity, as described further below. Inaddition, the FMCP proteins can be used to screen drugs or compoundswhich modulate the FMCP activity or expression as well as to treatdisorders characterized by insufficient or excessive production of FMCPprotein or production of FMCP protein forms which have decreased oraberrant activity compared to FMCP wild type protein (e.g. proliferativedisorders such as cancer or preclampsia). In addition, the anti-FMCPantibodies of the invention can be used to detect and isolate FMCPproteins and modulate FMCP activity.

This invention further pertains to novel agents identified by the abovedescribed screening assays and uses thereof for treatments as describedherein.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to FMCP proteins or have a stimulatory or inhibitory effecton, for example, FMCP expression or FMCP activity.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of themembrane-bound form of a FMCP protein or polypeptide or biologicallyactive portion thereof. The test compounds of the present invention canbe obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of FMCP protein, or a biologicallyactive portion thereof, on the cell surface is contacted with a testcompound and the ability of the test compound to bind to a FMCP proteindetermined. The cell, for example, can of mammalian origin or a yeastcell. Determining the ability of the test compound to bind to the FMCPprotein can be accomplished, for example, by coupling the test compoundwith a radioisotope or enzymatic label such that binding of the testcompound to the FMCP protein or biologically active portion thereof canbe determined by detecting the labeled compound in a complex. Forexample, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemission or by scintillation counting. Alternatively,test compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product. In a preferred embodiment, the assaycomprises contacting a cell which expresses a membrane-bound form ofFMCP protein, or a biologically active portion thereof, on the cellsurface with a known compound which binds FMCP to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a FMCP protein, whereindetermining the ability of the test compound to interact with a FMCPprotein comprises determining the ability of the test compound topreferentially bind to FMCP or a biologically active portion thereof ascompared to the known compound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of FMCP protein, or abiologically active portion thereof, on the cell surface with a testcompound and determining the ability of the test compound to modulate(e.g. stimulate or inhibit) the activity of the FMCP protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of FMCP or a biologically activeportion thereof can be accomplished, for example, by determining theability of the FMCP protein to bind to or interact with a FMCP targetmolecule. As used herein, a “target molecule” is a molecule with which aFMCP protein binds or interacts in nature, for example, a molecule onthe surface of a cell which expresses a FMCP protein, a molecule on thesurface of a second cell, a molecule in the extracellular milieu, amolecule associated with the internal surface of a cell membrane or acytoplasmic molecule. A FMCP target molecule can be a non-FMCP moleculeor a FMCP protein or polypeptide of the present invention. In oneembodiment, a FMCP target molecule is a component of a signaltransduction pathway which facilitates transduction of an extracellularsignal (e.g. a signal generated by binding of a compound to amembrane-bound FMCP molecule) through the cell membrane and into thecell. The target, for example, can be a second intercellular proteinwhich has catalytic activity or a protein which facilitates theassociation of downstream signaling molecules with FMCP.

Determining the ability of the FMCP protein to bind to or interact witha FMCP target molecule can be accomplished by one of the methodsdescribed above for determining direct binding. In a preferredembodiment, determining the ability of the FMCP protein to bind to orinteract with a FMCP target molecule can be accomplished by determiningthe activity of the target molecule. For example, the activity of thetarget molecule can be determined by detecting induction of a cellularsecond messenger of the target (i.e. intracellular Ca²⁺, diacylglycerol,IP₃, etc.), detecting catalytic/enzymatic activity of the target anappropriate substrate, detecting the induction of a reporter gene(comprising a FMCP-responsive regulatory element operatively linked to anucleic acid encoding a detectable marker, e.g. luciferase), ordetecting a cellular response, for example, cell survival, cellulardifferentiation, or cell proliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a FMCP protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the FMCP protein or biologically activeportion thereof. Binding of the test compound to the FMCP protein can bedetermined either directly or indirectly as described above. In apreferred embodiment, the assay comprises contacting the FMCP protein orbiologically active portion thereof with a known compound which bindsFMCP to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a FMCP protein, wherein determining the ability of the testcompound to interact with a FMCP protein comprises determining theability of the test compound to preferentially bind to FMCP orbiologically active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting FMCP protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g. stimulate or inhibit) the activity of the FMCP protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of FMCP can be accomplished, forexample, by determining the ability of the FMCP protein to bind to aFMCP target molecule by one of the methods described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of FMCP can beaccomplished by determining the ability of the FMCP protein furthermodulate a FMCP target molecule. For example, the catalytic/enzymaticactivity of the target molecule on an appropriate substrate can bedetermined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theFMCP protein or biologically active portion thereof with a knowncompound which binds FMCP to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with a FMCP protein, wherein determining theability of the test compound to interact with a FMCP protein comprisesdetermining the ability of the FMCP protein to preferentially bind to ormodulate the activity of a FMCP target molecule.

The cell-free assays of the present invention are amenable to use ofboth the soluble form or the membrane-bound form of FMCP. In the case ofcell-free assays comprising the membrane-bound form of FMCP, it may bedesirable to utilize a solubilizing such that the membrane-bound form ofFMCP is maintained in solution. Examples of such solubilizing agentsinclude non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either FMCP or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to FMCP, or interaction of FMCP with atarget molecule in the presence and absence of a candidate compound, canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/FMCP fusionproteins or glutathione-S-transferase/target fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or FMCP protein, and the mixture incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of FMCPbinding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either FMCP orits target molecule can be immobilized utilizing conjugation of biotinand streptavidin. Biotinylated FMCP or target molecules can be preparedfrom biotin-NHS (N-hydroxy-succinimide) using techniques well known inthe art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with FMCP or targetmolecules but which do not interfere with binding of the FMCP protein toits target molecule can be derivatized to the wells of the plate, andunbound target or FMCP trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the FMCP or target molecule, aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the FMCP or target molecule.

In another embodiment, modulators of FMCP expression are identified in amethod wherein a cell is contacted with a candidate compound and theexpression of FMCP mRNA or protein in the cell is determined. The levelof expression of FMCP mRNA or protein in the presence of the candidatecompound is compared to the level of expression of FMCP mRNA or proteinin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of FMCP expression based on thiscomparison. For example, when expression of FMCP mRNA or protein isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of FMCP mRNA or protein expression.Alternatively, when expression of FMCP mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of FMCP mRNA or protein expression. The level of FMCP mRNA orprotein expression in the cells can be determined by methods describedherein for detecting FMCP mRNA or protein.

In yet another aspect of the invention, the FMCP proteins can be used as“bait proteins” in a two-hybrid assay or three hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with FMCP (“FMCP-binding proteins” or “FMCP-bp”) and modulateFMCP activity. Such FMCP-binding proteins are also likely to be involvedin the propagation of signals by the FMCP proteins as, for example,upstream or downstream elements of the FMCP pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for FMCP is fused to agene encoding the DNA binding domain of a known transcription factor(e.g., GAL-4). In the other construct, a DNA sequence, from a library ofDNA sequences, that encodes an unidentified protein (“prey” or “sample”)is fused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming a FMCP-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g.,LacZ) which is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the protein which interacts with FMCP.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the FMCP, sequences, described herein, can beused to map the location of the FMCP genes, respectively, on achromosome. The mapping of the FMCP sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, FMCP genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the FMCP sequences.Computer analysis of the FMCP, sequences can be used to rapidly selectprimers that do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers can then be usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the FMCP sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the FMCPsequences to design oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes. Othermapping strategies which can similarly be used to map a 9o, 1p, or 1vsequence to its chromosome include in situ hybridization (described inFan, Y. et al. (1990) PNAS, 87:6223-27), pre-screening with labeledflow-sorted chromosomes, and pre-selection by hybridization tochromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the FMCP gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The FMCP sequences of the present invention can also be used to identifyindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the FMCP sequences described herein can be used to preparetwo PCR primers from the 5′ and 3′ ends of the sequences. These primerscan then be used to amplify an individual's DNA and subsequentlysequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The FMCP sequences of the invention uniquely represent portions of thehuman genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Each ofthe sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1 can comfortablyprovide positive individual identification with a panel of perhaps 10 to1,000 primers which each yield a noncoding amplified sequence of 100bases. If predicted coding sequences, such as those in SEQ ID NO:3 areused, a more appropriate number of primers for positive individualidentification would be 500-2,000.

If a panel of reagents from FMCP sequences described herein is used togenerate a unique identification database for an individual, those samereagents can later be used to identify tissue from that individual.Using the unique identification database, positive identification of theindividual, living or dead, can be made from extremely small tissuesamples.

3. Use of Partial FMCP Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NOs:1, 5, and 10 areparticularly appropriate for this use as greater numbers ofpolymorphisms occur in the noncoding regions, making it easier todifferentiate individuals using this technique. Examples ofpolynucleotide reagents include the FMCP sequences or portions thereof,e.g., fragments derived from the noncoding regions of SEQ ID NO:1,having a length of at least 20 bases, preferably at least 30 bases.

The FMCP sequences described herein can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes which can beused in, for example, an in situ hybridization technique, to identify aspecific tissue, e.g., brain tissue. This can be very useful in caseswhere a forensic pathologist is presented with a tissue of unknownorigin. Panels of such FMCP probes can be used to identify tissue byspecies and/or by organ type.

In a similar fashion, these reagents, e.g., FMCP primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningFMCP protein and/or nucleic acid expression as well as FMCP activity, inthe context of a biological sample (e.g., blood, serum, cells, tissue)to thereby determine whether an individual is afflicted with a diseaseor disorder, or is at risk of developing a disorder, associated withaberrant FMCP expression or activity. The invention also provides forprognostic (or predictive) assays for determining whether an individualis at risk of developing a disorder associated with FMCP protein,nucleic acid expression or activity. For example, mutations in a FMCPgene can be assayed in a biological sample. Such assays can be used forprognostic or predictive purpose to thereby phophylactically treat anindividual prior to the onset of a disorder characterized by orassociated with FMCP protein, nucleic acid expression or activity.

Another aspect of the invention provides methods for determining FMCPprotein, nucleic acid expression or FMCP activity in an individual tothereby select appropriate therapeutic or prophylactic agents for thatindividual (referred to herein as “pharmacogenomics”). Pharmacogenomicsallows for the selection of agents (e.g., drugs) for therapeutic orprophylactic treatment of an individual based on the genotype of theindividual (e.g., the genotype of the individual examined to determinethe ability of the individual to respond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs, compounds) on the expression or activity of FMCPin clinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of FMCP in abiological sample involves obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting FMCP protein or nucleic acid (e.g., mRNA, genomicDNA) that encodes FMCP protein such that the presence of FMCP isdetected in the biological sample. A preferred agent for detecting FMCPmRNA or genomic DNA is a labeled nucleic acid probe capable ofhybridizing to FMCP mRNA or genomic DNA. The nucleic acid probe can be,for example, a full-length FMCP nucleic acid, such as the nucleic acidof SEQ ID NO:1, or a portion thereof, such as an oligonucleotide of atleast 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficientto specifically hybridize under stringent conditions to FMCP mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

A preferred agent for detecting FMCP protein is an antibody capable ofbinding to FMCP protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect FMCP mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of FMCP mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of FMCP proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of FMCP genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of FMCP protein includeintroducing into a subject a labeled anti-FMCP antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting FMCP protein, mRNA, orgenomic DNA, such that the presence of FMCP protein, mRNA or genomic DNAis detected in the biological sample, and comparing the presence of FMCPprotein, mRNA or genomic DNA in the control sample with the presence ofFMCP protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of FMCPin a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting FMCP protein or mRNA in abiological sample; means for determining the amount of FMCP in thesample; and means for comparing the amount of FMCP in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectFMCP protein or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant FMCP expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with FMCP protein, nucleic acidexpression or activity such as cancer or fibrotic disorders.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing a disease or disorder. Thus,the present invention provides a method for identifying a disease ordisorder associated with aberrant FMCP expression or activity in which atest sample is obtained from a subject and FMCP protein or nucleic acid(e.g., mRNA, genomic DNA) is detected, wherein the presence of FMCPprotein or nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant FMCPexpression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant FMCP expression or activity. For example, suchmethods can be used to determine whether a subject can be effectivelytreated with an agent for a disorder, such as cancer or pre-clampsia.Thus, the present invention provides methods for determining whether asubject can be effectively treated with an agent for a disorderassociated with aberrant FMCP expression or activity in which a testsample is obtained and FMCP protein or nucleic acid is detected (e.g.,wherein the presence of FMCP protein or nucleic acid is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant FMCP expression or activity.)

The methods of the invention can also be used to detect genetic lesionsin a FMCP gene, thereby determining if a subject with the lesioned geneis at risk for a disorder characterized by aberrant cell proliferationand/or differentiation. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic lesion characterized by at least one of analteration affecting the integrity of a gene encoding a FMCP-protein, orthe mis-expression of the FMCP gene. For example, such genetic lesionscan be detected by ascertaining the existence of at least one of 1) adeletion of one or more nucleotides from a FMCP gene; 2) an addition ofone or more nucleotides to a FMCP gene; 3) a substitution of one or morenucleotides of a FMCP gene, 4) a chromosomal rearrangement of a FMCPgene; 5) an alteration in the level of a messenger RNA transcript of aFMCP gene, 6) aberrant modification of a FMCP gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a FMCP gene, 8) anon-wild type level of a FMCP-protein, 9) allelic loss of a FMCP gene,and 10) inappropriate post-translational modification of a FMCP-protein.As described herein, there are a large number of assay techniques knownin the art which can be used for detecting lesions in a FMCP gene. Apreferred biological sample is a peripheral blood leukocyte sampleisolated by conventional means from a subject.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the FMCP-gene (see Abravaya et al. (1995)Nucleic Acids Res. 23:675-682). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a FMCP gene under conditions such that hybridization andamplification of the FMCP-gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. It is anticipated that PCR and/or LCR may be desirable to use asa preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et all, 1988, Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a FMCP gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in FMCP can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in FMCP can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the FMCP gene anddetect mutations by comparing the sequence of the sample FMCP with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463). It is alsocontemplated that any of a variety of automated sequencing procedurescan be utilized when performing the diagnostic assays ((1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996)Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

Other methods for detecting mutations in the FMCP gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type FMCP sequence with potentially mutant RNAor DNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al (1988)Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control DNA or RNA can belabeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in FMCP cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a FMCP sequence,e.g., a wild-type FMCP sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in FMCP genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control FMCPnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985)Nature 313:495). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denature, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing gradient to identify differences in the mobility ofcontrol and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA86:6230). Such allele specific oligonucleotides are hybridized to PCRamplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a FMCP gene.

Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which FMCP is expressed may be utilized in the prognosticassays described herein.

3. Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect onFMCP activity (e.g., FMCP gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) disorders (e.g., cancer orgestational disorders) associated with aberrant FMCP activity. Inconjunction with such treatment, the pharmacogenomics (i.e., the studyof the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual's genotype. Suchpharmacogenomics can further be used to determine appropriate dosagesand therapeutic regimens. Accordingly, the activity of FMCP protein,expression of FMCP nucleic acid, or mutation content of FMCP genes in anindividual can be determined to thereby select appropriate agent(s) fortherapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See e.g., Eichelbaum, M., Clin Exp PharmacolPhysiol, 1996, 23(10-11):983-985 and Linder, M. W., Clin Chem, 1997,43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of FMCP protein, expression of FMCP nucleic acid, ormutation content of FMCP genes in an individual can be determined tothereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual. In addition, pharmacogenetic studies can beused to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a FMCP modulator, such as a modulator identified by one of theexemplary screening assays described herein.

4. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of FMCP (e.g., the ability to modulate aberrentcell proliferation and/or differentiation) can be applied not only inbasic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase FMCP gene expression, protein levels, or upregulateFMCP activity, can be monitored in clinical trails of subjectsexhibiting decreased FMCP gene expression, protein levels, ordownregulated FMCP activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease FMCP gene expression,protein levels, or downregulate FMCP activity, can be monitored inclinical trails of subjects exhibiting increased FMCP gene expression,protein levels, or upregulated FMCP activity. In such clinical trials,the expression or activity of FMCP and, preferably, other genes thathave been implicated in, for example, a cellular proliferation disordercan be used as a “read out” or markers of the immune responsiveness of aparticular cell.

For example, and not by way of limitation, genes, including FMCP, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates FMCP activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on cellular proliferation disorders, for example,in a clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of FMCP and other genes implicated in thedisorder. The levels of gene expression (i.e., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods as described herein, or by measuring thelevels of activity of FMCP or other genes. In this way, the geneexpression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,.nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a FMCP protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the FMCP protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the FMCP protein, mRNA, or genomic DNA inthe pre-administration sample with the FMCP protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of FMCP to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of FMCP to lower levels than detected, i.e. to decrease theeffectiveness of the agent.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder-associated with aberrant FMCP expression oractivity.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant FMCPexpression or activity, by administering to the subject an agent whichmodulates FMCP expression or at least one FMCP activity. Subjects atrisk for a disease which is caused or contributed to by aberrant FMCPexpression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a phophylactic agent can occur prior to themanifestation of symptoms characteristic of the FMCP aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of FMCP aberrancy, for example, aFMCP agonist or FMCP antagonist agent can be used for treating thesubject. The appropriate agent can be determined based on screeningassays described herein. The prophylactic methods of the presentinvention are further discussed in the following subsections.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating FMCPexpression or activity for therapeutic purposes. The modulatory methodof the invention involves contacting a cell with an agent that modulatesone or more of the activities of FMCP protein activity associated withthe cell. An agent that modulates FMCP protein activity can be an agentas described herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of a FMCP protein, a peptide, a FMCPpeptidomimetic, or other small molecule. In one embodiment, the agentstimulates one or more FMCP protein activity. Examples of suchstimulatory agents include active FMCP protein and a nucleic acidmolecule encoding FMCP that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more FMCP proteinactivity. Examples of such inhibitory agents include antisense FMCPnucleic acid molecules and anti-FMCP antibodies. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a FMCP protein or nucleic acidmolecule. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordownregulates) FMCP expression or activity. In another embodiment, themethod involves administering a FMCP protein or nucleic acid molecule astherapy to compensate for reduced or aberrant FMCP expression oractivity.

Stimulation of FMCP activity is desirable in situations in which FMCP isabnormally downregulated and/or in which increased FMCP activity islikely to have a beneficial effect. One example of such a situation iswhere a subject has a disorder characterized by aberrant cellproliferation and/or differentiation (e.g., cancer). Another example ofsuch a situation is where the subject has a gestational disease (e.g.,preclampsia).

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLES Example 1 Isolation and Characterization of Human FMCP cDNAs

In this example, the isolation of the genes encoding human FMCP isdescribed.

Isolation of FMCP

Human coronary artery smooth muscle cells (obtained from CloneticsCorporation; San Diego, Calif.) were expanded in culture with SmoothMuscle Growth Media (SmGM; Clonetics) according to the recommendationsof the supplier. When the cells reached 80% confluence, they werestimulated with SmGM, tumor necrosis factor (TNF—10 ng/ml) andcycloheximide (CHI; 40 micrograms/ml) for 4 hours. Total RNA wasisolated using the RNeasy Midi Kit (Qiagen; Chatsworth, Calif.), and thepoly A+ fraction was further purified using Oligotex beads (Qiagen).

Three micrograms of poly A+ RNA were used to synthesize a cDNA libraryusing the Superscript cDNA Synthesis kit (Gibco BRL; Gaithersburg, Md.).Complementary DNA was directionally cloned into the expression plasmidpMET7 using the SalI and NotI sites in the polylinker to construct aplasmid library. Transformants were picked and grown up for single-passsequencing. Additionally, coronary artery smooth muscle cDNA was ligatedinto the SalI/NotI sites of the ZipLox vector (Gibco BRL) forconstruction of a lambda phage cDNA library.

FMCP was identified using Sequence Explorer®, a sequence analysis toolthat integrates the output from high-throughput sequencing projects withautomated BLAST searches of the protein, nucleic and EST databases. Theoriginal first pass sequence of the FMCP clone showed homology to humanfollistatin using the BLASTX program, which translates a nucleic acidsequence in all six frames and compares it against available proteindatabases. This clone was then grown up for full sequencing to confirmthe homology to follistatin.

Example 2 Distribution of FMCP mRNA Human Tissues

The expression of FMCP was analyzed using Northern blot hybridization. A956 base pair (bp) DNA fragment (the SalI/BamHI fragment containing the5′ end of the FMCP cDNA) was used as a probe. The DNA was radioactivelylabeled with ³²P-dCTP using the Prime-It kit (Stratagene, La Jolla,Calif.) according to the instructions of the supplier. Filterscontaining human mRNA (MTNI and MTNII from Clontech, Palo Alto, Calif.)were probed in ExpressHyb hybridization solution (Clontech) and washedat high stringency according to manufacturer's recommendations.

FMCP is expressed as an ˜2.5 kilobase (kb) transcript in a wide varietyof tissues (heart, placenta, lung, liver, skeletal muscle, kidney,pancreas, spleen, prostate, testis, ovaries, small intestine, andcolon), with the highest levels found in the placenta, testis and heart.This is in good agreement with the size of the cDNA clone isolated. Inaddition, a smaller transcript of ˜1.4 kb is also seen in heart,placenta, lung, kidney and testis.

Example 3 Characterization of FMCP Proteins

In this example, the predicted amino acid sequences of the human FMCPproteins were compared to amino acid sequences of known proteins andvarious motifs were identified. In addition, the molecular weight of thehuman FMCP proteins was predicted.

The human FMCP cDNA encodes a protein of 263 amino acids (predicted MWof 25 kDa, not including post-translational modifications). A signalpeptide is predicted to exist from aa 1-26, using the prediction programSIGNALP (Henrik Nielsen, Jacob Engelbrecht, Soren Brunak and Gunnar vonHeijne “Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” (1997) Protein Engineering 10,1-6). The human protein appears to be secreted or retained in anintracellular compartment and there is no evidence of a transmembranedomain.

Alignment of the human FMCP protein with the human follistatin proteinusing the Wisconsin GCG sequence alignment program GAP, reveals thatFMCP is 43% identical and 61% similar to the human follistatin gene.Similarly, when FMCP is aligned with the human follistatin-related gene(hFRP) (Swiss Prot Q12841) using GAP, it show a 24% identity and 48%similarity to hFRP.

Example 4 Preparation of FMCP Protein

Recombinant FMCP can be produced in a variety of expression systems. Forexample, the mature FMCP peptide can be expressed as a recombinantglutathione-S-transferase (GST) fusion protein in E. coli and the fusionprotein can be isolated and characterized. Specifically, as describedabove, FMCP can be fused to GST and this fusion protein can be expressedin E. coli strain PEB 199. As FMCP is predicted to be 25 kD and GST ispredicted to be 26 kD, the fusion protein is predicted to be 51 kD inmolecular weight. Expression of the GST-FMCP fusion protein in PEB199can be induced with IPTG. The recombinant fusion protein can be purifiedfrom crude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the proteins purified from the bacteriallysates, the resultant fusion protein should be 51 kD in size.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

5 2525 base pairs nucleic acid single linear cDNA CDS 23..814 1CGCTGCCGTC TCTGCGTTCG CC ATG CGT CCC GGG GCG CCA GGG CCA CTC TGG 52 MetArg Pro Gly Ala Pro Gly Pro Leu Trp 1 5 10 CCT CTG CCC TGG GGG GCC CTGGCT TGG GCC GTG GGC TTC GTG AGC TCC 100 Pro Leu Pro Trp Gly Ala Leu AlaTrp Ala Val Gly Phe Val Ser Ser 15 20 25 ATG GGC TCG GGG AAC CCC GCG CCCGGT GGT GTT TGC TGG CTC CAG CAG 148 Met Gly Ser Gly Asn Pro Ala Pro GlyGly Val Cys Trp Leu Gln Gln 30 35 40 GGC CAG GAG GCC ACC TGC AGC CTG GTGCTC CAG ACT GAT GTC ACC CGG 196 Gly Gln Glu Ala Thr Cys Ser Leu Val LeuGln Thr Asp Val Thr Arg 45 50 55 GCC GAG TGC TGT GCC TCC GGC AAC ATT GACACC GCC TGG TCC AAC CTC 244 Ala Glu Cys Cys Ala Ser Gly Asn Ile Asp ThrAla Trp Ser Asn Leu 60 65 70 ACC CAC CCG GGG AAC AAG ATC AAC CTC CTC GGCTTC TTG GGC CTT GTC 292 Thr His Pro Gly Asn Lys Ile Asn Leu Leu Gly PheLeu Gly Leu Val 75 80 85 90 CAC TGC CTT CCC TGC AAA GAT TCG TGC GAC GGCGTG GAG TGC GGC CCG 340 His Cys Leu Pro Cys Lys Asp Ser Cys Asp Gly ValGlu Cys Gly Pro 95 100 105 GGC AAG GCG TGC CGC ATG CTG GGG GGC CGC CCGCGC TGC GAG TGC GCG 388 Gly Lys Ala Cys Arg Met Leu Gly Gly Arg Pro ArgCys Glu Cys Ala 110 115 120 CCC GAC TGC TCG GGG CTC CCG GCG CGG CTG CAGGTC TGC GGC TCA GAC 436 Pro Asp Cys Ser Gly Leu Pro Ala Arg Leu Gln ValCys Gly Ser Asp 125 130 135 GGC GCC ACC TAC CGC GAC GAG TGC GAG CTG CGCGCC GCG CGC TGC CGC 484 Gly Ala Thr Tyr Arg Asp Glu Cys Glu Leu Arg AlaAla Arg Cys Arg 140 145 150 GGC CAC CCG GAC CTG AGC GTC ATG TAC CGG GGCCGC TGC CGC AAG TCC 532 Gly His Pro Asp Leu Ser Val Met Tyr Arg Gly ArgCys Arg Lys Ser 155 160 165 170 TGT GAG CAC GTG GTG TGC CCG CGG CCA CAGTCG TGC GTC GTG GAC CAG 580 Cys Glu His Val Val Cys Pro Arg Pro Gln SerCys Val Val Asp Gln 175 180 185 ACG GGC AGC GCC CAC TGC GTG GTG TGT CGAGCG GCG CCC TGC CCT GTG 628 Thr Gly Ser Ala His Cys Val Val Cys Arg AlaAla Pro Cys Pro Val 190 195 200 CCC TCC AGC CCC GGC CAG GAG CTT TGC GGCAAC AAC AAC GTC ACC TAC 676 Pro Ser Ser Pro Gly Gln Glu Leu Cys Gly AsnAsn Asn Val Thr Tyr 205 210 215 ATC TCC TCG TGC CAC ATG CGC CAG GCC ACCTGC TTC CTG GGC CGC TCC 724 Ile Ser Ser Cys His Met Arg Gln Ala Thr CysPhe Leu Gly Arg Ser 220 225 230 ATC GGC GTG CGC CAC GCG GGC AGC TGC GCAGGC ACC CCT GAG GAG CCG 772 Ile Gly Val Arg His Ala Gly Ser Cys Ala GlyThr Pro Glu Glu Pro 235 240 245 250 CCA GGT GGT GAG TCT GCA GAA GAG GAAGAG AAC TTC GTG TGAGCCTGCA 821 Pro Gly Gly Glu Ser Ala Glu Glu Glu GluAsn Phe Val 255 260 GGACAGGCCT GGGCCTGGTG CCCGAGGCCC CCCATCATCCCCTGTTATTT ATTGCCACAG 881 CAGAGTCTAA TTTATATGCC ACGGACACTC CTTAGAGCCCGGATTCGGAC CACTTGGGGA 941 TCCCAGAACC TCCCTGACGA TATCCTGGAA GGACTGAGGAAGGGAGGCCT GGGGGCCGGC 1001 TGGTGGGTGG GATAGACCTG CGTTCCGGAC ACTGAGCGCCTGATTTAGGG CCCTTCTCTA 1061 GGATGCCCCA GCCCCTACCC TAAGACCTAT TGCCGGGGAGGATTCCACAC TTCCTCTCCT 1121 TTGGGGATAA ACCTATTAAT TATTGCTACT ATCAAGAGGGCTGGGCATTC TCTGCTGGTA 1181 ATTCCTGAAG AGGCATGACT GCTTTTCTCA GCCCCAAGCCTCTAGTCTGG GTGTGTACGG 1241 AGGGTCTAGC CTGGGTGTGT ACGGAGGGTC TAGCCTGGGTGAGTACGGAG GGTCTAGCCT 1301 GGGTGAGTAC GGAGGGTCTA GCCTGGGTGA GTACGGAGAGTCTAGCCTGG GTGTGTATGG 1361 AGGATCTAGC CTGGGTGAGT ATGGAGGGTC TAGCCTGGGTGAGTATGGAG GGTCTAGCCT 1421 GGGTGTGTAT GGAGGGTCTA GCCTGGGTGA GTATGGAGGGTCTAGCCTGG GTGTGTATGG 1481 AGGGTCTAGC CTGGGTGAGT ATGGAGGGTC TAGCCTGGGTGTGTACGGAG GGTCTAGTCT 1541 GAGTGCGTGT GGGGACCTCA GAACACTGTG ACCTTAGCCCAGCAAGCCAG GCCCTTCATG 1601 AAGGCCAAGA AGGCTGCCAC CATTCCCTGC CAGCCCAAGAACTCCAGCTT CCCCACTGCC 1661 TCTGTGTGCC CCTTTGCGTC CTGTGAAGGC CATTGAGAAATGCCCAGTGT GCCCCCTGGG 1721 AAAGGGCACG GCCTGTGCTC CTGACACGGG CTGTGCTTGGCCACAGAACC ACCCAGCGTC 1781 TCCCCTGCTG CTGTCCACGT CAGTTCATGA GGCAACGTCGCGTGGTCTCA GACGTGGAGC 1841 AGCCAGCGGC AGCTCAGAGC AGGGCACTGT GTCCGGCGGAGCCAAGTCCA CTCTGGGGGA 1901 GCTCTGGCGG GGACCACGGG CCACTGCTCA CCCACTGGCCCCGAGGGGGG TGTAGACGCC 1961 AAGACTCACG CATGTGTGAC ATCCAGAGTC CTGGAGCCGGGTGTCCCAGT GGCACCACTA 2021 GGTGCCTGCT GCCTCCACAG TGGGGTTCAC ACCCAGGGCTCCTTGGTCCC CCACAACCTG 2081 CCCCGGCCAG GCCTGCAGAC CCAGACTCCA GCCAGACCTGCCTCACCCAC CAATGCAGCC 2141 GGGGCTGGCG ACACCAGCCA GGTGCTGGTC TTGGGCCAGTTCTCCCACGA CGGCTCACCC 2201 TCCCCTCCAT CTGCGTTGAT GCTCAGAATC GCCTACCTGTGCCTGCGTGT AAACCACAGC 2261 CTCAGACCAG CTATGGGGAG AGGACAACAC GGAGGATATCCAGCTTCCCC GGTCTGGGGT 2321 GAGGAGTGTG GGGAGCTTGG GCATCCTCCT CCAGCCTCCTCCAGCCCCCA GGCAGTGCCT 2381 TACCTGTGGT GCCCAGAAAA GTGCCCCTAG GTTGGTGGGTCTACAGGAGC CTCAGCCAGG 2441 CAGCCCACCC CACCCTGGGG CCCTGCCTCA CCAAGGAAATAAAGACTCAA AGAAGCCAAA 2501 AAAAAAAAAA AAAAGGGCGG CCGC 2525 263 aminoacids amino acid linear protein 2 Met Arg Pro Gly Ala Pro Gly Pro LeuTrp Pro Leu Pro Trp Gly Ala 1 5 10 15 Leu Ala Trp Ala Val Gly Phe ValSer Ser Met Gly Ser Gly Asn Pro 20 25 30 Ala Pro Gly Gly Val Cys Trp LeuGln Gln Gly Gln Glu Ala Thr Cys 35 40 45 Ser Leu Val Leu Gln Thr Asp ValThr Arg Ala Glu Cys Cys Ala Ser 50 55 60 Gly Asn Ile Asp Thr Ala Trp SerAsn Leu Thr His Pro Gly Asn Lys 65 70 75 80 Ile Asn Leu Leu Gly Phe LeuGly Leu Val His Cys Leu Pro Cys Lys 85 90 95 Asp Ser Cys Asp Gly Val GluCys Gly Pro Gly Lys Ala Cys Arg Met 100 105 110 Leu Gly Gly Arg Pro ArgCys Glu Cys Ala Pro Asp Cys Ser Gly Leu 115 120 125 Pro Ala Arg Leu GlnVal Cys Gly Ser Asp Gly Ala Thr Tyr Arg Asp 130 135 140 Glu Cys Glu LeuArg Ala Ala Arg Cys Arg Gly His Pro Asp Leu Ser 145 150 155 160 Val MetTyr Arg Gly Arg Cys Arg Lys Ser Cys Glu His Val Val Cys 165 170 175 ProArg Pro Gln Ser Cys Val Val Asp Gln Thr Gly Ser Ala His Cys 180 185 190Val Val Cys Arg Ala Ala Pro Cys Pro Val Pro Ser Ser Pro Gly Gln 195 200205 Glu Leu Cys Gly Asn Asn Asn Val Thr Tyr Ile Ser Ser Cys His Met 210215 220 Arg Gln Ala Thr Cys Phe Leu Gly Arg Ser Ile Gly Val Arg His Ala225 230 235 240 Gly Ser Cys Ala Gly Thr Pro Glu Glu Pro Pro Gly Gly GluSer Ala 245 250 255 Glu Glu Glu Glu Asn Phe Val 260 771 base pairsnucleic acid single linear cDNA 3 ATGCGTCCCG GGGCGCCAGG GCCACTCTGGCCTCTGCCCT GGGGGGCCCT GGCTTGGGCC 60 GTGGGCTTCG TGAGCTCCAT GGGCTCGGGGAACCCCGCGC CCGGTGGTGT TTGCTGGCTC 120 CAGCAGGGCC AGGAGGCCAC CTGCAGCCTGGTGCTCCAGA CTGATGTCAC CCGGGCCGAG 180 TGCTGTGCCT CCGGCAACAT TGACACCGCCTGGTCCAACC TCACCCACCC GGGGAACAAG 240 ATCAACCTCC TCGGCTTCTT GGGCCTTGTCCACTGCCTTC CCTGCAAAGA TTCGTGCGAC 300 GGCGTGGAGT GCGGCCCGGG CAAGGCGTGCCGCATGCTGG GGGGCCGCCC GCGCTGCGAG 360 TGCGCGCCCG ACTGCTCGGG GCTCCCGGCGCGGCTGCAGG TCTGCGGCTC AGACGGCGCC 420 ACCTACCGCG ACGAGTGCGA GCTGCGCGCCGCGCGCTGCC GCGGCCACCC GGACCTGAGC 480 GTCATGTACC GGGGCCGCTG CCGCAAGTCCTGTGAGCACG TGGTGTGCCC GCGGCCACAG 540 TCGTGCGTCG TGGACCAGAC GGGCAGCGCCCACTGCGTGG TGTGTCGAGC GGCGCCCTGC 600 CCTGTGCCCT CCAGCCCCGG CCAGGAGCTTTGCGGCAACA ACAACGTCAC CTACATCTCC 660 TCGTGCCACA TGCGCCAGGC CACCTGCTTCCTGGGCCGCT CCATCGGCGT GCGCCACGCG 720 GGCAGCTGCG CAGGCACCCC TGAGGAGCCGCCAGGTGGTG AGTCTGCAGA A 771 71 amino acids amino acid linear peptideinternal 4 Asp Ser Cys Asp Gly Val Glu Cys Gly Pro Gly Lys Ala Cys ArgMet 1 5 10 15 Leu Gly Gly Arg Pro Arg Cys Glu Cys Ala Pro Asp Cys SerGly Leu 20 25 30 Pro Ala Arg Leu Gln Val Cys Gly Ser Asp Gly Ala Thr TyrArg Asp 35 40 45 Glu Cys Glu Leu Arg Ala Ala Arg Cys Arg Gly His Pro AspLeu Ser 50 55 60 Val Met Tyr Arg Gly Arg Cys 65 70 73 amino acids aminoacid linear peptide internal 5 Cys Glu His Val Val Cys Pro Arg Pro GlnSer Cys Val Val Asp Gln 1 5 10 15 Thr Gly Ser Ala His Cys Val Val CysArg Ala Ala Pro Cys Pro Val 20 25 30 Pro Ser Ser Pro Gly Gln Glu Leu CysGly Asn Asn Asn Val Thr Tyr 35 40 45 Ile Ser Ser Cys His Met Arg Gln AlaThr Cys Phe Leu Gly Arg Ser 50 55 60 Ile Gly Val Arg His Ala Gly Ser Cys65 70

What is claimed:
 1. An isolated polypeptide comprising an amino acidsequence which is at least 55% identical to the amino acid sequence ofSEQ ID NO:2 with or without the signal peptide or an amino acid sequenceencoded by the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number
 98546. 2. The isolatedpolypeptide of claim 1, which comprises an amino acid sequence which isat least 75% identical to the amino acid sequence of SEQ ID NO:2 with orwithout the signal peptide or an amino acid sequence encoded by thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number
 98546. 3. The isolated polypeptide of claim 2, whichcomprises an amino acid sequence which is at least 85% identical to theamino acid sequence of SEQ ID NO:2 with or without the signal peptide oran amino acid sequence encoded by the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number
 98546. 4.The isolated polypeptide of claim 3, which comprises an amino acidsequence which is at least 95% identical to the amino acid sequence ofSEQ ID NO:2 with or without the signal peptide or an amino acid sequenceencoded by the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number
 98546. 5. The isolatedpolypeptide of claim 1, 2, 3, or 4 wherein the percent identity iscalculated using the Wisconsin GCG sequence alignment program GAP. 6.The isolated polypeptide of claim 1, 2, 3, or 4 wherein the polypeptidecomprises an amino acid sequence which is at least 85% identical to afollistatin cysteine-rich domain selected from the group consisting ofthe follistatin cysteine-rich domain comprising amino acid residues 97to 167 of SEQ ID NO:2 and the follistatin cysteine-rich domaincomprising amino acid residues 171 to 243 of SEQ ID NO:2.
 7. Theisolated polypeptide of claim 6, wherein the follistatin cysteine-richdomain comprises amino acid residues 97 to 167 of SEQ ID NO:2.
 8. Theisolated polypeptide of claim 6, wherein the follistatin cysteine-richdomain comprises amino acid residues 171 to 243 of SEQ ID NO:2.
 9. Theisolated polypeptide of claim 1, 2, 3, or 4, wherein the polypeptidecomprises an amino acid sequence which is at least 85% identical to thefollistatin cysteine-rich domain comprising amino acid residues 97 to167 of SEQ ID NO:2 and the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2.
 10. The isolatedpolypeptide of claim 9, wherein the polypeptide comprises thefollistatin cysteine-rich domain comprising amino acid residues 97 to167 of SEQ ID NO:2 and the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2.
 11. An isolatedpolypeptide comprising an amino acid sequence at least about 55%identical to a follistatin-cysteine-rich domain of SEQ ID NO:2.
 12. Theisolated polypeptide of claim 11, wherein the amino acid sequence is atleast about 65% identical to a follistatin-cysteine-rich domain of SEQID NO:2.
 13. The isolated polypeptide of claim 12, wherein the aminoacid sequence is at least about 75% identical to afollistatin-cysteine-rich domain of SEQ ID NO:2.
 14. The isolatedpolypeptide of claim 13, wherein the amino acid sequence is at leastabout 85% identical to a follistatin-cysteine-rich domain of SEQ IDNO:2.
 15. The isolated polypeptide of claim 11, 12, 13, or 14, whereinthe follistatin cysteine-rich domain comprises amino acid residues 97 to167 of SEQ ID NO:2.
 16. The isolated polypeptide of claim 11, 12, 13, or14, wherein the follistatin cysteine-rich domain comprises amino acidresidues 171 to 243 of SEQ ID NO:2.
 17. An isolated polypeptidecomprising the follistatin cysteine-rich domain comprising amino acidresidues 97 to 167 of SEQ ID NO:2 and the follistatin cysteine-richdomain comprising amino acid residues 171 to 243 of SEQ ID NO:2.
 18. Anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:2.19. An isolated polypeptide comprising at least 15 consecutive aminoacid residues of the amino acid sequence of SEQ ID NO:2 or the aminoacid sequence encoded by the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number
 98564. 20. Theisolated polypeptide of claim 19 which comprises at least 20 consecutiveamino acid residues of the amino acid sequence of SEQ ID NO:2 or theamino acid sequence encoded by the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number
 98564. 21. Theisolated polypeptide of claim 20 which comprises at least 30 consecutiveamino acid residues of the amino acid sequence of SEQ ID NO:2 or theamino acid sequence encoded by the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number
 98564. 22. Anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:2without the signal peptide.
 23. An isolated polypeptide comprising theamino acid sequence encoded by the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number
 98546. 24. Anisolated mature polypeptide comprising the amino acid sequence encodedby the nucleotide sequence of the DNA insert of the plasmid depositedwith ATCC as Accession Number
 98546. 25. An isolated polypeptidecomprising an amino acid sequence which is at least 85% identical to thefollistatin cysteine-rich domain comprising amino acid residues 97 to167 of SEQ ID NO:2 or the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2, wherein the polypeptideis encoded by a nucleic acid molecule comprising a nucleotide sequencewhich hybridizes under stringent hybridization conditions to thenucleotide sequence of SEQ ID NO:1.
 26. The isolated polypeptide ofclaim 25 comprising the follistatin cysteine-rich domain comprisingamino acid residues 97 to 167 of SEQ ID NO:2 or the follistatincysteine-rich domain comprising amino acid residues 171 to 243 of SEQ IDNO:2.
 27. An isolated polypeptide comprising an amino acid sequencewhich is at least 85% identical to the follistatin cysteine-rich domaincomprising amino acid residues 97 to 167 of SEQ ID NO:2 or thefollistatin cysteine-rich domain comprising amino acid residues 171 to243 of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleotidesequence which hybridizes under stringent hybridization conditions tothe nucleotide sequence of SEQ ID NO:3.
 28. The isolated polypeptide ofclaim 27, wherein the polypeptide comprises the follistatincysteine-rich domain comprising amino acid residues 97 to 167 of SEQ IDNO:2 or the follistatin cysteine-rich domain comprising amino acidresidues 171 to 243 of SEQ ID NO:2.
 29. An isolated polypeptidecomprising an amino acid sequence which is at least 85% identical to thefollistatin cysteine-rich domain comprising amino acid residues 97 to167 of SEQ ID NO:2 or the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2, wherein the polypeptideis encoded by a nucleotide sequence which hybridizes under stringenthybridization conditions to the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number
 98546. 30. Theisolated polypeptide of claim 29, wherein the polypeptide comprises thefollistatin cysteine-rich domain comprising amino acid residues 97 to167 of SEQ ID NO:2 or the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2.
 31. An isolatedpolypeptide comprising the follistatin cysteine-rich domain comprisingamino acid residues 97 to 167 of SEQ ID NO:2 and the follistatincysteine-rich domain comprising amino acid residues 171 to 243 of SEQ IDNO:2, wherein the polypeptide is encoded by a nucleotide sequence whichhybridizes under stringent hybridization conditions to the nucleotidesequence of SEQ ID NO:1.
 32. An isolated polypeptide comprising thefollistatin cysteine-rich domains comprising amino acid residues 97 to167 of SEQ ID NO:2 and the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2, wherein the polypeptideis encoded by a nucleotide sequence which hybridizes under stringenthybridization conditions to the nucleotide sequence of SEQ ID NO:3. 33.An isolated polypeptide comprising the follistatin cysteine-rich domaincomprising amino acid residues 97 to 167 of SEQ ID NO:2 and thefollistatin cysteine-rich domain comprising amino acid residues 171 to243 of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleotidesequence which hybridizes under stringent hybridization conditions tothe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number
 98546. 34. An isolated polypeptide comprisingthe follistatin cysteine-rich domain comprising amino acid residues 97to 167 of SEQ ID NO:2 or the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2, wherein the polypeptideis encoded by a nucleic acid molecule which hybridizes under stringenthybridization conditions to a nucleic acid molecule comprisingnucleotides 311 to 751 of SEQ ID NO:1.
 35. An isolated polypeptideencoded by a nucleic acid molecule which hybridizes under stringenthybridization conditions to a nucleic acid molecule comprisingnucleotides 311 to 751 of SEQ ID NO:1.
 36. An isolated polypeptideencoded by a nucleic acid molecule which hybridizes under stringenthybridization conditions to a nucleic acid molecule comprisingnucleotides 311 to 523 of SEQ ID NO:1.
 37. An isolated polypeptideencoded by a nucleic acid molecule which hybridizes under stringenthybridization conditions to a nucleic acid molecule comprisingnucleotides 533 to 751 of SEQ ID NO:1.
 38. An isolated polypeptideencoded by a nucleic acid molecule comprising a nucleotide sequencewhich hybridizes under stringent hybridization conditions to thenucleotide sequence of SEQ ID NO:1.
 39. An isolated polypeptide encodedby a nucleic acid molecule comprising a nucleotide sequence whichhybridizes under stringent hybridization conditions to the nucleotidesequence of SEQ ID NO:3.
 40. An isolated polypeptide encoded by anucleic acid molecule comprising a nucleotide sequence which hybridizesunder stringent hybridization conditions to the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number98546.
 41. An isolated polypeptide encoded by a nucleic acid molecule atleast 500 nucleotides in length which hybridizes under stringenthybridization conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1.
 42. An isolated polypeptide encodedby a nucleic acid molecule at least 500 nucleotides in length whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:3.
 43. Anisolated polypeptide encoded by a nucleic acid molecule at least 500nucleotides in length which hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98546.
 44. The isolated polypeptide of claim 41, 42, or 43, whichincludes the follistatin cysteine-rich domain comprising amino acidresidues 97 to 167 of SEQ ID NO:2 or the follistatin cysteine-richdomain comprising amino acid residues 171 to 243 of SEQ ID NO:2.
 45. Anisolated polypeptide containing a follistatin cysteine-rich domain,wherein the polypeptide is encoded by a nucleotide sequence which is atleast 55% identical to the nucleotide sequence of SEQ ID NO:1, SEQ IDNO:3, or the DNA insert of the plasmid deposited with ATCC as AccessionNumber
 98546. 46. The isolated polypeptide of claim 45, wherein thenucleotide sequence is at least 75% identical to the nucleotide sequenceof SEQ ID NO:1, SEQ ID NO:3, or the DNA insert of the plasmid depositedwith ATCC as Accession Number
 98546. 47. The isolated polypeptide ofclaim 46, wherein the nucleotide sequence is at least 85% identical tothe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the DNA insertof the plasmid deposited with ATCC as Accession Number
 98546. 48. Theisolated polypeptide of claim 47, wherein the nucleotide sequence is atleast 95% identical to the nucleotide sequence of SEQ ID NO:1, SEQ IDNO:3, or the DNA insert of the plasmid deposited with ATCC as AccessionNumber
 98546. 49. The isolated polypeptide of claim 45, 46, 47, or 48,wherein the percent identity is calculated using the Wisconsin GCGsequence alignment program GAP.
 50. The isolated polypeptide of claim45, 46, 47, or 48, wherein the polypeptide comprises an amino acidsequence which is at least 85% identical to the follistatincysteine-rich domain selected from the group consisting of thefollistatin-cysteine-rich domain comprising amino acid residues 97 to167 of SEQ ID NO:2 and the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2.
 51. The isolatedpolypeptide of claim 50, wherein the polypeptide comprises thefollistatin cysteine-rich domain comprising amino acid residues 97 to167 of SEQ ID NO:2 or the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2.
 52. The isolatedpolypeptide of claim 45, 46, 47, or 48, wherein the polypeptidecomprises an amino acid sequence which is at least 85% identical to thefollistatin cysteine-rich domain comprising amino acid residues 97 to167 of SEQ ID NO:2 and the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2.
 53. The isolatedpolypeptide of claim 52, wherein the polypeptide comprises thefollistatin cysteine-rich domain comprising amino acid residues 97 to167 of SEQ ID NO:2 and the follistatin cysteine-rich domain comprisingamino acid residues 171 to 243 of SEQ ID NO:2.
 54. An isolatedpolypeptide encoded by a nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:1.
 55. An isolated polypeptide encoded by anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3.56. An isolated polypeptide encoded by a nucleic acid moleculecomprising the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number
 98546. 57. An isolatedpolypeptide consisting of the amino acid sequence of SEQ ID NO:2.
 58. Anisolated polypeptide encoded by a nucleic acid molecule consisting ofthe nucleotide sequence of SEQ ID NO:1.
 59. An isolated polypeptideencoded by a nucleic acid molecule consisting of the nucleotide sequenceof SEQ ID NO:3.
 60. An isolated polypeptide encoded by nucleic acidmolecule consisting of the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number
 98546. 61. The isolatedpolypeptide of claim 1 or 19 further comprising, an amino acid sequencecorresponding to a protein which is not substantially homologous to theamino acid sequence of SEQ ID NO:2.